dCas9-SPO11-1 locally stimulates meiotic recombination in rice

Abstract

Introduction: Meiotic crossovers shuffle the genetic information transmitted by the gametes. However, the potential to recover all the combinations of the parental alleles remains limited in most organisms, including plants, by the occurrence of only few crossovers per chromosome and a prominent bias in their spatial distribution. Thus, novel methods for stimulating recombination frequencies and/or modifying their location are highly desired to accelerate plant breeding.

Methods: Here, we investigate the use of a dCas9-SPO11-1 fusion and clusters of 11 gRNAs to alter meiotic recombination in two chromosomal regions of a rice hybrid (KalingaIII/Kitaake). To accurately genotype rare recombinants in regions of few kbp, we improved the digital PCR-based pollen-typing method in parallel.

Results: Expression of the dCas9-SPO11-1 fusion protein under the ubiquitous ZmUbi1 promoter was obtained in leaves/anthers/meiocytes and found to complement the sterility of the Osspo11-1 mutant line. We observed a 3.27-fold increase over wild-type (p<0.001) of recombinant pollens in a transgenic hybrid line (7a) targeting a chromosome 7 region. In the offspring plant 7a1, a significant 2.05-fold increase (p=0.048) was observed in the central interval (7.2 kb) of the Chr. 7 target region. This stimulation of meiotic recombination is consistent with the expression of the dCas9-SPO11-1 fusion and gRNAs as well as with the ChIP-revealed binding of dCas9-SPO11-1 to the targeted region. In contrast, no stimulation was observed in other transgenic lines deficient in the above pre-requisite features, expressing the dCas9-SPO11-1 fusion but no gRNAs or targeting a Chr.9 region.

Discussion: These results open new avenues to locally stimulate meiotic recombination in crop genomes and paves the way for a future implementation in plant breeding programs.

Keywords: CRISPR/dCas9; Spo11; meiosis; plant breeding; pollen typing; rice; targeted recombination.

Figure 1

Induction of double-strand breaks by dCas9-SPO11-1 and gRNAs transgenes. (A) Schematic representation of hypothetical DNA repair following dCas9-SPO11-1-induced Double Strand Break (DSB) at prophase of meiosis. Left: dCas9 (gray), fused to the SPO11-1 subunit (light blue), binds the target site and recruits the other components of the transesterase Spo11 complex, including SPO11-2 (orange) and MTOPVIB (grey). Right: Recombinational repair of Spo11-dependent DSB in meiosis: Spo11-dependent DSB formation on one of the homologous chromosomes (dark) and ssDNA resection allow strand invasion of the homologous chromosome. Progression and resolution of the recombination intermediates yield recombined non-crossover (NCO) and crossover (CO) molecules adapted from Yelina et al. (2022). (B) Structure of the T-DNA constructs co-introduced in rice. Up: T-DNA for dCas9-OsSPO11-1 expression. Down: T-DNA for transcription of the multiplexed tRNA:gRNA scaffold, which once managed by the natural tRNA processing system is supposed to release 11 gRNAs targeting either Chr.7 or Chr.9 regions (gRNAs and tRNAs are shown as purple and black boxes, respectively).

Figure 2

Molecular and functional characterization of the transgenic plants 7a and 7b. (A) Schematic representation of the recombination context of the targeted Chr.7 region established by whole genome sequencing of a 379 F2 progeny population of KalingaIII/Kitaake (Unpublished). The position of the dPCR (7#1, 7#2, 7#3 and 7#4) and Kasp (Z1 to Z4) markers with their coordinates on the Kitaake genome are detailed on the zoom. Kasp markers ZA to ZD were only used to genotype plant 7a offsprings ( Figure 5 ). The green bar represents the area containing the recombinant identified in the progeny. The sequence of the polymorphic markers and 11 gRNAs (pink bars) are given in Supplementary Tables S4 , S9 . The 11 gRNAs target the 5’ promoter region of the expansin-like A3 gene (OsKitaake07g138900.1). (B) RT-qPCR quantification of the dCas9 and SPO11-1 transcripts in leaf tissues of the transgenic KalingaIII/Kitaake plants 7a and 7b relative to OsKitaake07g010600.1 Expressed protein gene as reference (n=2). Values follow a Log10 scale. (C) dCas9-SPO11-1 accumulation in leaf tissues of plants 7a and 7b revealed by western blot analysis using an anti-V5 antibody. Expected gel migration positions of dCas9 (161 kDa) and dCas9-SPO11-1 (206 kDa) are pointed by blue and red arrowheads, respectively. (D) RT-qPCR accumulation of the gRNAs in leaf tissues of transgenic plants 7a and 7b. Values follow a Log10 scale and use the OsKitaake07g010600.1 gene as a reference (n=2). (E) ChIP-qPCR of the targeted chromosome 7 region. Chromatin DNA of WT KalingaIII and Kitaake parents and independent transgenic KalingaIII/Kitaake hybrid plants 7a and 7b were immuno-precipitated using the anti-v5 antibody and quantified by qPCR. The sonicated fragments are in the range of 200-900 bp. Target sequences of the 11 gRNA scaffold are shown in purple. The qPCR amplified regions (area 1 and 2) are indicated by red arrowheads. The enrichment values are normalized by the input (10% of total chromatin). The 3’ region of the ubiquitously expressed gene OsKitaake06g078500.1 residing on Chr.6 was used as a non-target control (n=3).

Figure 3

Meiotic expression and functional complementation of Osspo11-1 by the dCas9-SPO11-1 fusion. (A) Recovery of fertile T0 plants regenerated from Osspo11-/- seed embryo-derived calluses harboring the dCas9-OsgSPO11-1 (genomic SPO11-1 DNA) driven by the maize ubiquitin 1 (pZmUbi1) promoter region T-DNA (3 independent events) or a dCas9-empty, the T-DNA devoid of the gSPO11-1 sequence (7 independent events) or the dCas9-cSPO11-1 (OsSPO11-1 cDNA) T-DNA (5 independent events). Only dCas9-OsgSPO11-1 T-DNA restores fertility in Osspo11-1-1 -/- segregant progeny plants. (B) Fertility assessment of complemented Osspo11-1-1 mutant lines. The spo11-1-1, WT and heterozygous WT/spo11-1-1 genotypes contain both plants from pure lines and plants from complemented lines that have segregated the dCas9-empty, dCas9-gDNASPO11-1 or dCas9cDNASPO11-1 T-DNA. The dCas9-SPO11-1 individuals come from 2 independent fixed T3 lines and are all homozygous for the spo11-1-1 mutation. Means are compared using a T.test between the WT and the other groups (p > 0.01**; p > 0.001***). (C) Photograph of WT, spo11-1-1 mutant and complemented rice panicles used for fertility assessments in B (Scale bar=1cm). (D) RT-qPCR quantification of two dCas9-SPO11-1 spo11-1 kitaake mutant plants transcripts in anthers and meiocytes tissues relative to OsKitaake07g010600.1 Expressed protein gene as reference. Amplifications were performed using primers located on the interval between the end of dCas9 and SPO11-1 including the linker. Values follow a Log10 scale. (E) dCas9-SPO11-1 accumulation in leaf tissues of plants 7a1 (used as positive control, see Figure 6 ), dCas9-empty plant and in anther tissues of two dCas9-gDNASPO11-1-complemented spo11-1 plants revealed by western blot analysis using an anti-V5 antibody. Expected gel migration positions of dCas9 (161 kDa) and dCas9-SPO11-1 (206 kDa) are pointed by blue and red arrowheads, respectively. In contrast to the leaf tissues of plant 7a1, we only detect the dCas9-SPO11-1 protein in its integral form in anther tissues.

Figure 4

QIAcuity Digital PCR pollen typing strategy. (A) Experimental setup of the pollen typing experiment using dPCR. (B) Outline of the recombination assay. Left: The probes are distributed in a staggered pattern along the Kitaake and KalingaIII target chromosomal regions. Right: Probe associations are diagnostic of parental and recombinant alleles. (C) Optimization of the dPCR assay. After FACS sorting, a better cluster dissociation was observed between positive (blue) and negative (gray) partitions (threshold: red line) when nuclei are loaded in dPCR mix completed with H₂O rather than with the LB01 buffer. (D) Fraction of genotyped nuclei (2 probes) per well when 1,000 or 2,000 nuclei are loaded. The volume of loaded nuclei through a 70 µm nozzle in the dPCR mix is 1.7µL and 3.4µL for 1,000 and 2,000 nuclei respectively. (E) Comparison of the specificities of chromosome 7 probes 7#1 and 7#2 specificity. Each sample of 1,000 nuclei is represented as a 1D Scatterplot for each probe and their fluorescence intensity (RFU). Each point represents a partition. A restrictive threshold (red line) separates gray (negative) and blue (positive) partitions. Each positive partition shows a fluorescence association that allows the nucleus genotype to be determined. Wells are also checked in 2D scatterplots (associations between 2 probes) to refine threshold positioning. Here no clear signal dissociation is observed for probe 7#1 between the KalingaIII and Kitaake alleles. The threshold (red line) signal dissociation is improved when 1,000 nuclei are loaded. In contrast, the signal of the probe 7#2 (as well as the probes 7#3 and 7#4, data not shown) clearly distinguishes the KalingaIII and Kitaake alleles. (additional information in Methods and Supplementary Figure S6 ).

Figure 5

Recombination frequencies in the dCas9-SPO11-1 events and controls. (A) Positions of the KalingaIII and Kitaake markers flanking the targeted Chr.7 region. Probes are designed using the SNP/Indels shown in the table. Physical distances are given after the first gRNA position (17 005 820 on the Kitaake Chr.7). (B, C) Recombination frequencies over the target region of chromosome 7 deduced from genotyping nuclei WT and dCas9-SPO11-1 plants 7a and 7b pollen. Values are corrected by removing the background (Methods). Frequencies are compared over the whole region and then over each interval. The detailed data set is reported in Table 1 . The corrected number of recombinant nuclei is indicated under recombination rate values. Fisher exact test was performed between WT and plants 7a and 7b (p values are shown, p>0.05*; p>0.01**; p>0.001***, ns, not significant).

Figure 6

Molecular and functional characterization of the transgenic offspring plants 7a1 and 7a2. (A) RT-qPCR quantification of the dCas9 and SPO11-1 transcripts in leaf tissues of the transgenic plants 7a1 and 7a2 relative to OsKitaake07g010600.1 Expressed protein gene as reference (n=3). Values follow a Log10 scale. (B) RT-qPCR accumulation of the gRNAs in leaf tissues of transgenic plants 7a1 and 7a2. Values follow a Log10 scale and use the Kitaake Os07g010600.1 gene as a reference. (C) dCas9-SPO11-1 accumulation in leaf tissues of plants 7a1 and 7a2 revealed by western blot analysis using an anti-V5 antibody. Expected gel migration positions of dCas9 (161 kDa) and dCas9-SPO11-1 (206 kDa) are pointed by blue and red arrowheads, respectively. (D) ChIP-qPCR of the targeted chromosome 7 region. Chromatin DNA of WT hybrid and offspring of the hybrid plants 7a1 and 7a2 were immuno-precipitated using the anti-v5 antibody and quantified by qPCR. The sonicated fragments are in the range of 200-900 bp. Target sequences of the 11 gRNA scaffold are shown in purple. The qPCR amplified regions (area 1 and 2) are indicated by red arrowheads. The enrichment values are normalized by the input (10% of total chromatin). The 3’ region of the ubiquitously expressed gene OsKitaake06g078500.1 residing on Chr.6 was used as a non-target control (n=3). (E) Recombination frequencies over the target region of chromosome 7 deduced from genotyping nuclei WT and dCas9-SPO11-1 plant 7a1 or 7a2 pollen. Values are corrected by removing the background (Methods). Frequencies are compared over the whole region and then over each interval. The detailed data set is reported in Table 1 . The corrected number of recombinant nuclei is indicated under recombination rate values. Fisher exact test was performed between WT and plant 7a1 or 7a2 (p values are shown, p>0.05*, ns, not significant).